Structures of the Substrate-free and Product-bound Forms of HmuO,a Heme Oxygenase from Corynebacterium diphtheriae: X-RAY CRYSTALLOGRAPHY AND MOLECULAR DYNAMICS INVESTIGATION*?

Heme oxygenase catalyzes the degradation of heme to biliverdin, iron, and carbon monoxide. Here, we present crystal structures of the substrate-free, Fe³⁺-biliverdin-bound, and biliverdin-bound forms of HmuO, a heme oxygenase from Corynebacterium diphtheriae, refined to 1.80, 1.90, and 1.85 Å resolution, respectively. In the substrate-free structure, the proximal and distal helices, which tightly bracket the substrate heme in the substrate-bound heme complex, move apart, and the proximal helix is partially unwound. These features are supported by the molecular dynamic simulations. The structure implies that the heme binding fixes the enzyme active site structure, including the water hydrogen bond network critical for heme degradation. The biliverdin groups assume the helical conformation and are located in the heme pocket in the crystal structures of the Fe³⁺-biliverdin-bound and the biliverdin-bound HmuO, prepared by in situ heme oxygenase reaction from the heme complex crystals. The proximal His serves as the Fe³⁺-biliverdin axial ligand in the former complex and forms a hydrogen bond through a bridging water molecule with the biliverdin pyrrole nitrogen atoms in the latter complex. In both structures, salt bridges between one of the biliverdin propionate groups and the Arg and Lys residues further stabilize biliverdin at the HmuO heme pocket. Additionally, the crystal structure of a mixture of two intermediates between the Fe³⁺-biliverdin and biliverdin complexes has been determined at 1.70 Å resolution, implying a possible route for iron exit.     

IruO Is a Reductase for Heme Degradation by IsdI and IsdG Proteins in Staphylococcus aureus

Staphylococcus aureus is a common hospital- and community-acquired bacterium that can cause devastating infections and is often multidrug-resistant. Iron acquisition is required by S. aureus during an infection, and iron acquisition pathways are potential targets for therapies. The gene NWMN2274 in S. aureus strain Newman is annotated as an oxidoreductase of the diverse pyridine nucleotide-disulfide oxidoreductase (PNDO) family. We show that NWMN2274 is an electron donor to IsdG and IsdI catalyzing the degradation of heme, and we have renamed this protein IruO. Recombinant IruO is a FAD-containing NADPH-dependent reductase. In the presence of NADPH and IruO, either IsdI or IsdG degraded bound heme 10-fold more rapidly than with the chemical reductant ascorbic acid. Varying IsdI-heme substrate and monitoring loss of the heme Soret band gave a K_m of 15 ± 4 μm, a k_cat of 5.2 ± 0.7 min⁻¹, and a k_cat/K_m of 5.8 × 10³ m⁻¹ s⁻¹. From HPLC and electronic spectra, the major heme degradation products are 5-oxo-δ-bilirubin and 15-oxo-β-bilirubin (staphylobilins), as observed with ascorbic acid. Although heme degradation by IsdI or IsdG can occur in the presence of H₂O₂, the addition of catalase and superoxide dismutase did not disrupt NADPH/IruO heme degradation reactions. The degree of electron coupling between IruO and IsdI or IsdG remains to be determined. Homologs of IruO were identified by sequence similarity in the genomes of Gram-positive bacteria that possess IsdG-family heme oxygenases. A phylogeny of these homologs identifies a distinct clade of pyridine nucleotide-disulfide oxidoreductases likely involved in iron uptake systems. IruO is the likely in vivo reductant required for heme degradation by S. aureus.     

Roles of distal Asp in heme oxygenase from Corynebacterium diphtheriae, HmuO: A water-driven oxygen activation mechanism

Heme oxygenases found in mammals, plants, and bacteria catalyze degradation of heme using the same mechanism. Roles of distal Asp (Asp-136) residue in HmuO, a heme oxygenase of Corynebacterium diphtheriae, have been investigated by site-directed mutagenesis, enzyme kinetics, resonance Raman spectroscopy, and x-ray crystallography. Replacements of the Asp-136 by Ala and Phe resulted in reduced heme degradation activity due to the formation of ferryl heme, showing that the distal Asp is critical in HmuO heme oxygenase activity. D136N HmuO catalyzed heme degradation at a similar efficiency to wild type and D136E HmuO, implying that the carboxylate moiety is not required for the heme catabolism by HmuO. Resonance Raman results suggest that the inactive ferryl heme formation in the HmuO mutants is induced by disruption of the interaction between a reactive Fe-OOH species and an adjacent distal pocket water molecule. Crystal structural analysis of the HmuO mutants confirms partial disappearance of this nearby water in D136A HmuO. Our results provide the first experimental evidence for the catalytic importance of the nearby water molecule that can be universally critical in heme oxygenase catalysis and propose that the distal Asp helps in positioning the key water molecule at a position suitable for efficient activation of the Fe-OOH species.     

HmuS and HmuQ of <Emphasis Type="Italic">Ensifer</Emphasis>/<Emphasis Type="Italic">Sinorhizobium meliloti</Emphasis> degrade heme in vitro and participate in heme metabolism in vivo

Ensifer meliloti is a nitrogen-fixing symbiont of the alfalfa legume able to use heme as an iron source. The transport mechanism involved in heme acquisition in E. meliloti has been identified and characterized, but the fate of heme once inside the cell is not known. In silico analysis of E. meliloti 1021 genome revealed no canonical heme oxygenases although two genes encoding putative heme degrading enzymes, smc01518 and hmuS, were identified. SMc01518 is similar to HmuQ of Bradyrhizobium japonicum, which is weakly homologous to the Staphylococcus aureus IsdG heme-degrading monooxygenase, whereas HmuS is homolog to Pseudomonas aeruginosa PhuS, a protein reported as a heme chaperone and as a heme degrading enzyme. Recombinant HmuQ and HmuS were able to bind hemin with a 1:1 stoichiometry and displayed a K_d value of 5 and 4 µM, respectively. HmuS degrades heme in vitro to the biliverdin isomers IX-β and IX-δ in an equimolar ratio. The HmuQ recombinant protein degrades heme to biliverdin IX-δ only. Additionally, in this work we demonstrate that humS and hmuQ gene expression is regulated by iron and heme in a RirA dependent manner and that both proteins are involved in heme metabolism in E. meliloti in vivo.     

Structure of the Escherichia coli O157:H7 heme oxygenase ChuS in complex with heme and enzymatic inactivation by mutation of the heme coordinating residue His-193

Heme oxygenases catalyze the oxidation of heme to biliverdin, CO, and free iron. For pathogenic microorganisms, heme uptake and degradation are critical mechanisms for iron acquisition that enable multiplication and survival within hosts they invade. Here we report the first crystal structure of the pathogenic Escherichia coli O157:H7 heme oxygenase ChuS in complex with heme at 1.45 A resolution. When compared with other heme oxygenases, ChuS has a unique fold, including structural repeats and a beta-sheet core. Not surprisingly, the mode of heme coordination by ChuS is also distinct, whereby heme is largely stabilized by residues from the C-terminal domain, assisted by a distant arginine from the N-terminal domain. Upon heme binding, there is no large conformational change beyond the fine tuning of a key histidine (His-193) residue. Most intriguingly, in contrast to other heme oxygenases, the propionic side chains of heme are orientated toward the protein core, exposing the alpha-meso carbon position where O(2) is added during heme degradation. This unique orientation may facilitate presentation to an electron donor, explaining the significantly reduced concentration of ascorbic acid needed for the reaction. Based on the ChuS-heme structure, we converted the histidine residue responsible for axial coordination of the heme group to an asparagine residue (H193N), as well as converting a second histidine to an alanine residue (H73A) for comparison purposes. We employed spectral analysis and CO measurement by gas chromatography to analyze catalysis by ChuS, H193N, and H73A, demonstrating that His-193 is the key residue for the heme-degrading activity of ChuS.     

The heme complex of Hmu O, a bacterial heme degradation enzyme from Corynebacterium diphtheriae. Structure of the catalytic site.

Hmu O, a heme degradation enzyme in Corynebacterium diphtheriae, forms a stoichiometric complex with iron protoporphyrin IX and catalyzes the oxygen-dependent conversion of hemin to biliverdin, carbon monoxide, and free iron. Using a multitude of spectroscopic techniques, we have determined the axial ligand coordination of the heme-Hmu O complex. The ferric complex shows a pH-dependent reversible transition between a water-bound hexacoordinate high spin neutral pH form and an alkaline form, having high spin and low spin states, with a pK(a) of 9. (1)H NMR, EPR, and resonance Raman of the heme-Hmu O complex establish that a neutral imidazole of a histidine residue is the proximal ligand of the complex, similar to mammalian heme oxygenase. EPR of the deoxy cobalt porphyrin IX-Hmu O complex confirms this proximal histidine coordination. Oxy cobalt-Hmu O EPR reveals a hydrogen-bonding interaction between the O(2) and an exchangeable proton in the Hmu O distal pocket and two distinct orientations for the bound O(2). Mammalian heme oxygenase has only one O(2) orientation. This difference and the mixed spin states at alkaline pH indicate structural differences in the distal environment between Hmu O and its mammalian counterpart.     

Crystal structure of HugZ, a novel heme oxygenase from Helicobacter pylori

The crystal structure of a heme oxygenase (HO) HugZ from Helicobacter pylori complexed with heme has been solved and refined at 1.8 Å resolution. HugZ is part of the iron acquisition mechanism of H. pylori, a major pathogen of human gastroenteric diseases. It is required for the adaptive colonization of H. pylori in hosts. Here, we report that HugZ is distinct from all other characterized HOs. It exists as a dimer in solution and in crystals, and the dimer adopts a split-barrel fold that is often found in FMN-binding proteins but has not been observed in hemoproteins. The heme is located at the intermonomer interface and is bound by both monomers. The heme iron is coordinated by the side chain of His²⁴⁵ and an azide molecule when it is present in crystallization conditions. Experiments show that Arg¹⁶⁶, which is involved in azide binding, is essential for HugZ enzymatic activity, whereas His²⁴⁵, surprisingly, is not, implying that HugZ has an enzymatic mechanism distinct from other HOs. The placement of the azide corroborates the observed γ-meso specificity for the heme degradation reaction, in contrast to most known HOs that have α-meso specificity. We demonstrate through sequence and structural comparisons that HugZ belongs to a new heme-binding protein family with a split-barrel fold. Members of this family are widespread in pathogenic bacteria and may play important roles in the iron acquisition of these bacteria.     

Mutagenesis of nitrite reductase from Pseudomonas aeruginosa: tyrosine-10 in the c heme domain is not involved in catalysis

In Pseudomonas aeruginosa, conversion of nitrite to NO in dissimilatory denitrification is catalyzed by the enzyme nitrite reductase (NiR), a homodimer containing a covalently bound c heme and a d₁ heme per subunit. We report the purification and characterization of the first single mutant of P. aeruginosa cd₁ NiR in which Tyr¹⁰ has been replaced by Phe; this amino acid was chosen as a possibly important residue in the catalytic mechanism of this enzyme based on the proposal (Fülöp, V., Moir, J.W.B., Ferguson, S.J. and Hajdu, J. (1995) Cell 81, 369–377) that the topologically homologous Tyr²⁵ plays a crucial role in controlling the activity of the cd₁ NiR from Thiosphaera pantotropha. Our results show that in P. aeruginosa NiR substitution of Tyr¹⁰ with Phe has no effect on the activity, optical spectroscopy and electron transfer kinetics of the enzyme, indicating that distal coordination of the Fe³⁺ of the d₁ heme is provided by different side-chains in different species.     

l-Glyceraldehyde 3-phosphate reductase from Escherichia coli is a heme binding protein

Recently, we reported that YghZ from Escherichia coli functions as an efficient l-glyceraldehyde 3-phosphate reductase (Gpr). Here we show that Gpr co-purifies with a b-type heme cofactor. Gpr associates with heme in a 1:1 stoichiometry to form a complex that is characterized by a K_d value of 5.8 ± 0.2 μM in the absence of NADPH and a K_d value of 11 ± 1.3 μM in the presence of saturating NADPH. The absorbance spectrum of reconstituted Gpr indicates that heme is bound in a hexacoordinate low-spin state under both oxidizing and reducing conditions. The physiological function of heme association with Gpr is unclear, as the l-glyceraldehyde 3-phosphate reductase activity of Gpr does not require the presence of the cofactor. Bioinformatics analysis reveals that Gpr clusters with a family of putative monooxygenases in several organisms, suggesting that Gpr may act as a heme-dependent monooxygenase. The discovery that Gpr associates with heme is interesting because Gpr shares 35% amino acid identity with the mammalian voltage-gated K⁺ channel β-subunit, an NADPH-dependent oxidoreductase that endows certain voltage-gated K⁺ channels with hemoprotein-like, O₂-sensing properties. To date the molecular origin of O₂ sensing by voltage-gated K⁺ channels is unknown and the results presented herein suggest a role for heme in this process.     

Structure of cytochrome c551 from Pseudomonas aeruginosa refined at 1.6 Å resolution and comparison of the two redox forms

The molecular structures of ferri- and ferrocytochrome c₅₅₁ from Pseudomonas aeruginosa have been refined at a resolution of 1.6 Å, to an R factor of 19.5% for the oxidized molecule and 18.7% for the reduced. Reduction of oxidized crystals with ascorbate produced little change in cell dimensions, a 10% mean change in F_obs, and no damage to the crystals. The heme iron is not significantly displaced from the porphyrin plane. Bond lengths from axial ligands to the heme iron are as expected in a low-spin iron compound. A total of 67 solvent molecules were incorporated in the oxidized structure, and 73 in the reduced, of which four are found inside the protein molecule. The oxidized and reduced forms have virtually identical tertiary structures with 2 ° root-mean-square differences in main-chain torsion angles φ and ψ, but with larger differences along the two edges of the heme crevice. The difference map and pyrrole ring tilt suggest that a partially buried water molecule (no. 23) in the heme crevice moves upon change of oxidation state.Pseudomonas cytochrome c₅₅₁ differs from tuna cytochrome c in having: (1) a water molecule (no. 23) at the upper left of the heme crevice; that is, between Pro62 and the heme pyrrol 3 ring on the sixth ligand Met61 side, where tuna cytochrome c has an evolutionary invariant Phe82 ring; (2) a string of hydrophobic side-chains along the left side of the heme crevice, and fewer positively charged lysines in the vicinity; and (3) a more exposed and presumably more easily ionizable heme propionate group at the bottom of the molecule. A network of hydrogen bonds in the heme crevice is reminiscent of that inside the heme crevice of tuna cytochrome c. As in tuna, a slight motion of the water molecule toward the heme is observed in the oxidized state, helping to give the heme a more polar microenvironment. The continuity of solvent environment between the heme crevice and the outer medium could explain the greater dependence of redox potential on pH in cytochrome c₅₅₁ than in cytochrome c.     

Spectroscopic characterization of a higher plant heme oxygenase isoform-1 from Glycine max (soybean)--coordination structure of the heme complex and catabolism of heme

Heme oxygenase converts heme into biliverdin, CO, and free iron. In plants, as well as in cyanobacteria, heme oxygenase plays a particular role in the biosynthesis of photoreceptive pigments, such as phytochromobilins and phycobilins, supplying biliverdin IX(alpha) as a direct synthetic resource. In this study, a higher plant heme oxygenase, GmHO-1, of Glycine max (soybean), was prepared to evaluate the molecular features of its heme complex, the enzymatic activity, and the mechanism of heme conversion. The similarity in the amino acid sequence between GmHO-1 and heme oxygenases from other biological species is low, and GmHO-1 binds heme with 1 : 1 stoichiometry at His30; this position does not correspond to the proximal histidine of other heme oxygenases in their sequence alignments. The heme bound to GmHO-1, in the ferric high-spin state, exhibits an acid-base transition and is converted to biliverdin IX(alpha) in the presence of NADPH/ferredoxin reductase/ferredoxin, or ascorbate. During the heme conversion, an intermediate with an absorption maximum different from that of typical verdoheme-heme oxygenase or CO-verdoheme-heme oxygenase complexes was observed and was extracted as a bis-imidazole complex; it was identified as verdoheme. A myoglobin mutant, H64L, with high CO affinity trapped CO produced during the heme degradation. Thus, the mechanism of heme degradation by GmHO-1 appears to be similar to that of known heme oxygenases, despite the low sequence homology. The heme conversion by GmHO-1 is as fast as that by SynHO-1 in the presence of NADPH/ferredoxin reductase/ferredoxin, thereby suggesting that the latter is the physiologic electron-donating system.     

The ferrous–dioxy complex of Leishmania major globin coupled heme containing adenylate cyclase: The role of proximal histidine on its stability

Recently we have described the globin-coupled heme containing adenylate cyclase from Leishmania major (HemAC-Lm) that shows an O₂ dependent cAMP signaling (Sen Santara, et. al. Proc. Natl. Acad. Sci. U.S.A. 110, 16790–16795 (2013)). The heme iron of HemAC-Lm is expected to participate in oxygen binding and activates adenylate cyclase activity during catalysis, but its interactions with O₂ are uncharacterized. We have utilized the HemAC-Lm and stopped-flow methods to study the formation and decay of the HemAC-Lm oxygenated complex at 25 °C. Mixing of the ferrous HemAC-Lm with air-saturated buffer generates a very stable oxygenated complex with absorption maxima at 414, 540 and 576 nm. The distal axial ligand in the deoxygenated ferrous HemAC-Lm is displaced by O₂ at a rate of ~ 10 s^− 1. To prepare apoprotein of heme iron in HemAC-Lm, we have mutated the proximal His161 to Ala and characterized the mutant protein. The apo as well as heme reconstituted ferric state of the mutant protein shows a ~ 30 fold lower catalytic activity compared to oxygenated form of wild type protein. The oxygenated form of heme reconstituted mutant protein is highly unstable (decay rate = 6.1 s^− 1). Decomposition of the oxygenated intermediate is independent of O₂ concentration and is monophasic. Thus, the stabilization of ferrous-oxy species is an essential requirement in the wild type HemAC-Lm for a conformational alteration in the sensor domain that, sequentially, activates the adenylate cyclase domain, resulting in the synthesis of cAMP.     

Stability properties of sperm whale oxymyoglobin

Sperm whale oxymyoglobin was isolated directly from muscle and was examined for its stability properties over the wide range of pH 5–13 in 0.1 m buffer at 25 °C. The remarkable pH dependence for the autoxidation rate was analyzed using the kinetic equation derived in terms of nucleophilic displacement processes of O₂^? from oxymyoglobin by the entering water molecule or hydroxyl ion with the iron resulting in the ferric form. Most of the autoxidation reaction of the oxymyoglobin can be best explained by the proton-catalyzed processes involving the distal histidine as the catalytic residue. The kinetic equation could also be used as an interesting diagnostic probe into differences in the heme reactivity and the heme environment of different types of oxymyoglobin from other sources.     

The Soret magnetic circular dichroism of ferric high-spin myoglobins. A probe for the distal histidine residue.

To find a simple criterion for the presence of the distal (E7) histidine residue in myoglobins and hemoglobins, the Soret magnetic-circular-dichroic spectra were examined for ferric metmyoglobins from various species. A distinct and symmetric dispersion-type curve was obtained for myoglobins containing the distal histidine, whereas a relatively weak and unsymmetric pattern was observed for myoglobins lacking this residue, such as those from three kinds of gastropodic sea molluscs, a shark and the African elephant. The magnetic-circular-dichroic spectra obtained would thus be a direct reflection of the presence or absence of a water molecule at the sixth coordinate position of the heme iron(III), this axial water ligand being stabilized by hydrogen-bond formation to the distal histidine residue. On the basis of these Soret magnetic-circular-dichroic signals, we also examined the structure of a protozoan myoglobin (or a monomeric hemoglobin) from Paramecium caudatum of particular interest for the evolution of these proteins from protozoa to higher animals.     

Crystallographic structure of Rhodospirillum molischianum ferricytochrome c' at 2.5 A resolution

This paper describes the 2.5 Å crystallographic structure determination of ferricytochrome c′ from the photosynthetic bacterium Rhodospirillum molischianum. The molecule is a symmetric dimer, with each 128-residue polypeptide chain incorporating a covalently bound protoheme IX prosthetic group. The monomer is structurally organized as an array of four nearly parallel α-helices, which pack most closely at one end and thereafter spatially diverge to accommodate the heme prosthetic group. Although local features of the heme attachment pattern resemble those seen in cytochrome c, the heme iron in cytochrome c′ is pentaco-ordinate with a solvent-exposed histidine residue furnishing the single axial ligand to the heme iron.Subunit association in the dimeric molecule is principally stabilized by helix interactions, which are qualitatively similar to those occurring within each monomer. These interactions result in a dimer geometry that situates the exposed regions of both hemes on the same molecular surface.The structural basis for some of the physiochemical properties cytochrome c′ are examined and compared to those of other heme proteins of known structure.     

Staphylococcus lugdunensis IsdG Liberates Iron from Host Heme

Staphylococcus lugdunensis is often found as part of the normal flora of human skin but has the potential to cause serious infections even in healthy individuals. It remains unclear what factors enable S. lugdunensis to transition from a skin commensal to an invasive pathogen. Analysis of the complete genome reveals a putative iron-regulated surface determinant (Isd) system encoded within S. lugdunensis. In other bacteria, the Isd system permits the utilization of host heme as a source of nutrient iron to facilitate bacterial growth during infection. In this study, we establish that S. lugdunensis expresses an iron-regulated IsdG-family heme oxygenase that binds and degrades heme. Heme degradation by IsdG results in the release of free iron and the production of the chromophore staphylobilin. IsdG-mediated heme catabolism enables the use of heme as a sole source of iron, establishing IsdG as a pathophysiologically relevant heme oxygenase in S. lugdunensis. Together these findings offer insight into how S. lugdunensis fulfills its nutritional requirements while invading host tissues and establish the S. lugdunensis Isd system as being involved in heme-iron utilization.     

Axial coordination of heme in ferric CcmE chaperone characterized by EPR spectroscopy

In Escherichia coli cytochrome c maturation requires a set of eight proteins including the heme chaperone CcmE, which binds heme transiently, yet covalently. Several variants of CcmE were purified and analyzed by continuous-wave electron paramagnetic resonance, electron nuclear double resonance, and hyperfine sublevel correlation spectroscopy to investigate the heme axial coordination. Results reveal the presence of a number of coordination environments, two high-spin heme centers with different rhombicities, and at least one low-spin heme center. The low-spin species was shown to be an artifact induced by the presence of available histidines in the vicinity of the iron. Both of the high-spin forms are five-coordinated, and comparison of the spectra of the wild-type CcmE with those of the mutant CcmE(Y134H) proves that the higher-rhombicity form is coordinated by Tyr134. The low-rhombicity (axial) form does not have a histidine residue or a water molecule as an axial ligand. However, we identified exchangeable protons coupled to the iron ion. We propose that the axial form can be coordinated by a carboxyl group of an acidic residue in the flexible domain of the protein. The two species would represent two different conformations of the flexible alpha-helix domain surrounding the heme. This conformational flexibility confers CcmE special dynamic properties that are certainly important for its function.     

Cytochromes: Reactivity of the "dark side" of the heme

Ligand binding to the heme distal side is a paradigm of heme-protein biochemistry, the proximal axial ligand being in most cases a His residue. NO binds to the ferrous heme-Fe-atom giving rise to hexa-coordinated adducts (as in myoglobin and hemoglobin) with His and NO as proximal and distal axial ligands, respectively, or to penta-coordinated adducts (as in soluble guanylate cyclase) with NO as the axial distal ligand. Recently, the ferrous derivative of Alcaligenes xylosoxidans cytochrome c' (Axcyt c') and of cardiolipin-bound horse heart cytochrome c (CL-hhcyt c) have been reported to bind NO to the "dark side" of the heme (i.e., as the proximal axial ligand) replacing the endogenous ligand His. Conversely, CL-free hhcyt c behaves as ferrous myoglobin by binding NO to the heme distal side, keeping His as the proximal axial ligand. Moreover, the ferrous derivative of CL-hhcyt c binds CO at the heme distal side, the proximal axial ligand being His. Furthermore, CL-hhcyt c shows peroxidase activity. In contrast, CL-free hhcyt c does not bind CO and does not show peroxidase activity. This suggests that heme-proteins may utilize both sides of the heme for ligand discrimination, which appears to be modulated allosterically. Here, structural and functional aspects of NO binding to ferrous Axcyt c' and (CL-)hhcyt c are reviewed.     

Structural Analysis and Identification of PhuS as a Heme-Degrading Enzyme from Pseudomonas aeruginosa

Bacterial pathogens require iron for proliferation and pathogenesis. Pseudomonas aeruginosa is a prevalent Gram-negative opportunistic human pathogen that takes advantage of immunocompromised hosts and encodes a number of proteins for uptake and utilization of iron. Here we report the crystal structures of PhuS, previously known as the cytoplasmic heme-trafficking protein from P. aeruginosa, in both the apo- and the holo-forms. In comparison to its homologue ChuS from Escherichia coli O157:H7, the heme orientation is rotated 180° across the α-γ axis, which may account for some of the unique functional properties of PhuS. In contrast to previous findings, heme binding does not result in an overall conformational change of PhuS. We employed spectroscopic analysis and CO measurement by gas chromatography to analyze heme degradation, demonstrating that PhuS is capable of degrading heme using ascorbic acid or cytochrome P450 reductase-NADPH as an electron donor and produces five times more CO than ChuS. Addition of catalase slows down but does not stop PhuS-catalyzed heme degradation. Through spectroscopic and mass spectrometry analysis, we identified the enzymatic product of heme degradation to be verdoheme. These data taken together suggest that PhuS is a potent heme-degrading enzyme, in addition to its proposed heme-trafficking function.     

Direct Tests of Enzymatic Heme Degradation by the Malaria Parasite Plasmodium falciparum

Malaria parasites generate vast quantities of heme during blood stage infection via hemoglobin digestion and limited de novo biosynthesis, but it remains unclear if parasites metabolize heme for utilization or disposal. Recent in vitro experiments with a heme oxygenase (HO)-like protein from Plasmodium falciparum suggested that parasites may enzymatically degrade some heme to the canonical HO product, biliverdin (BV), or its downstream metabolite, bilirubin (BR). To directly test for BV and BR production by P. falciparum parasites, we DMSO-extracted equal numbers of infected and uninfected erythrocytes and developed a sensitive LC-MS/MS assay to quantify these tetrapyrroles. We found comparable low levels of BV and BR in both samples, suggesting the absence of HO activity in parasites. We further tested live parasites by targeted expression of a fluorescent BV-binding protein within the parasite cytosol, mitochondrion, and plant-like plastid. This probe could detect exogenously added BV but gave no signal indicative of endogenous BV production within parasites. Finally, we recombinantly expressed and tested the proposed heme degrading activity of the HO-like protein, PfHO. Although PfHO bound heme and protoporphyrin IX with modest affinity, it did not catalyze heme degradation in vivo within bacteria or in vitro in UV absorbance and HPLC assays. These observations are consistent with PfHO''s lack of a heme-coordinating His residue and suggest an alternative function within parasites. We conclude that P. falciparum parasites lack a canonical HO pathway for heme degradation and thus rely fully on alternative mechanisms for heme detoxification and iron acquisition during blood stage infection.